Location: Cell Wall Biology and Utilization Research
2023 Annual Report
Objectives
Objective 1: Develop or adapt poly-phenol systems in forage legumes for improved N use efficiency in dairy production systems.
Subobjective 1.1: Evaluate efficacy of the polyphenol oxidase (PPO)/o-diphenol system on preserving true protein during ensiling and improving N-use efficiency.
Subobjective 1.2: Determine the chemical basis for proteolytic inhibition caused by PPO-generated o-quinones.
Subobjective 1.3: Develop strategies to produce optimal levels of PPO substrates in alfalfa.
Objective 2: Develop or adapt tannin systems in forage legumes for improved N use efficiency in dairy production systems.
Subobjective 2.1: Determine the chemical basis for protection of protein during rumen digestion and providing elevated levels of escape protein into the hindgut by condensed tannins (CTs).
Subobjective 2.2: Analyze the effects of harvesting and storage methods on active CT content and protein preservation.
Objective 3: Improve forage digestibility and nutrient utilization efficiency in the cow through physiological modifications.
Subobjective 3.1: Prevent excessive leaf loss during plant development and harvesting by identifying genetic factors involved in leaf abscission in alfalfa providing a foundation for gene-based strategies for improvement.
Subobjective 3.2: Use genetic manipulation of sugar nucleotide biosynthetic pathways to identify avenues for altering cell wall structural polysaccharides and matrix interactions.
Subobjective 3.3: Explore alfalfa physiological mechanisms to enhance the utility of alfalfa as a cattle feed and other uses.
Objective 4: Improve forage silage quality and preservation to lessen forage losses, improve nutrition value for the cow, enhance soil ecology, and reduce environmental impacts for integrated dairy systems.
Subobjective 4.1: Incorporate non-traditional silage additives (novel forages, inoculants, concentrates) and management strategies to reduce forage loss and improve relative nutrition in the animal.
Subobjective 4.2: Leverage computational and sequencing technologies to elucidate connections between plant, silo, and animal microbiomes.
Objective 5: Develop system-based models to assess the productivity, efficiency, and environmental impact of dairy forage production.
Develop a whole-farm dairy simulation model that can be used to assess the impact of forage crop modifications and management on farm-scale nutrient cycling, farm crop and milk productivity, and environmental impacts.
Approach
Will utilize a multidisciplinary approach combining plant physiology/biochemistry, chemistry, agronomy, microbiology, molecular biology and genetics, and computer modeling. Forages provide unique nutritional and environmental opportunities to improve sustainable farming systems that help ensure food security. To enhance positive characteristics of forages, work will focus on capturing more plant protein in products, i.e., milk and plant bio-products, while generating less nitrogen waste; improving the amount of digestible cell wall biomass; and developing approaches to best maintain and optimize nutritional quality after harvest and during storage. We will also evaluate impacts of forage improvements and management by whole farm modeling. Efficient capture of protein nitrogen in the rumen is related to slowing protein degradation and availability of adequate digestible carbohydrate. Molecular, chemical, and biochemical approaches will be used to determine the roles of polyphenol oxidase/o-diphenols and tannins in decreasing protein degradation during ensiling and in the rumen (Objectives 1 and 2). Molecular approaches will be used to introduce a polyphenol oxidase/o-diphenol system into alfalfa to protect proteins during ensiling, including optimizing biochemical pathways in alfalfa to produce the o-diphenol PPO substrates. Chemical characterization of polyphenol (e.g., o-quinones and tannins) interactions with proteins will reveal mechanisms to protect proteins from degradation and provide selection criterion for forage improvement. Multiple approaches will be used to improve production of digestible forage biomass (especially carbohydrate) for improved animal performance (Objective 3). Molecular approaches will be used to down-regulate leaf abscission genes which would prevent excessive leaf loss, preserving a highly digestible fraction of alfalfa. The role of sugar nucleotide biosynthetic pathways in cell wall assembly and their influence on digestibility will be evaluated using molecular biology and biochemistry techniques. Approaches to maintain and optimize nutrition of preserved forages will be investigated using engineering, microbiological, and genomic approaches (Objective 4). Novel silage additives to prevent nutrient losses (for example via volatile organic compounds) will be investigated at lab and farm scales; microbial selection will be used to improve silage fermentation profiles, which could have impacts on greenhouse gas emissions; and metagenomics will be used to examine the complex interactions of field, silo, and rumen microbiomes. In order to better assess how changes in forages and forage management/storage impact the whole farm/agroecosystem, better whole-farm computer models will be developed with collaborators inside and outside of ARS (Objective 5). This project plan will increase our knowledge and understanding of current limitations associated with forage utilization and provides avenues to overcome these limitations.
Progress Report
For Objectives 1 and 2, we have examined two natural systems that have potential to improve nitrogen (N)-use efficiency in dairy production. Reducing protein losses just 10% could save U.S. farmers $200 to 400 million annually and reduce release of excess N into the environment. We previously identified a system of protein protection in red clover consisting of the enzyme polyphenol oxidase (PPO) and PPO-oxidizable o-diphenols. Adapting the clover system to forages like alfalfa requires providing both components, either by physical addition or by genetic modification of forages. We carried out small scale ensiling experiments with alfalfa expressing the PPO gene where PPO substrate was exogenously applied. Total N, non-protein N, and other silage quality parameters from these samples were analyzed. Unfortunately, in most instances no significant reductions in protein degradation were seen due to PPO and o-diphenols in these experiments, in contrast to original proof-of-concept experiments. The failure to detect protein protection in the current experiments may be due to the method of tissue maceration and additional experimental variables that increased variability between silos and ensiling conditions. In a different experiment, transgenic alfalfa plants with the PPO trait were crossed with transgenic alfalfa with the o-diphenol trait to reconstruct the complete system. Approximately 200 plants were screened for the traits, and several were identified expressing both PPO and producing the PPO substrate phaselic acid. Unfortunately, levels of phaselic acid produced were lower than the original parental plants, possibly due to genetic background. We examined proteolysis in extracts of these plants, but could not detect significant reductions compared to wild type plants in contrast to a previous experiment in which the system was reconstituted by mixing extracts. We believe failure to detect proteolytic reduction is due to inadequate levels of phaselic acid in these plants such that any effect cannot be detected over experimental variability. This experiment will be repeated to confirm the results.
Thus, for Sub-objective 1.3, we continued work on optimizing/increasing production of o-diphenol PPO substrates in alfalfa. We previously identified an enzyme and its gene (HMT [hydroxycinnamoyl-CoA:malate transferase]) involved in making one of the major o-diphenolic compounds in red clover, caffeoyl-malate, but HMT expression in alfalfa lead to accumulation of related compounds p-coumaroyl- and feruloyl-malate. Simultaneous downregulation of endogenous caffeoyl-CoA O-methyltransferase (CCOMT) resulted in drastically increased caffeoyl-malate levels. We prepared additional plant transformation constructs to enhance expression of phenylpropanoid pathway enzymes responsible for conversion of p-coumarate to caffeate. We have successfully modified our alfalfa transformation protocol for the phosphinothricin (Basta) resistance selectable marker used for these constructs and anticipate producing plants and being able to assess the impact of these additional genetic modifications on phaselic acid accumulation. To investigate how structure of transferases like HMT affect their function, we have been making detailed kinetic measurements of several transferases and a collaborator is determining structure by x-ray crystallography. Our collaborator has recently been able to determine structure for HMT which may provide strategies to improve o-diphenol production by this class of enzyme.
For Sub-objective 2.1, we continue multiple approaches to investigate the potential benefits of condensed tannin- (CT-) containing forages in animal production systems. This includes characterization of CT structure, biochemistry, and bioactivities. Development of 2D (two-dimensional) NMR (nuclear magnetic resonance) techniques to determine composition/structural features of purified CTs, which allows determination of how CT structure impacts biological activity, is near completion. This approach provides the same structural information as more conventional, but labor intensive, thiolytic degradation and hydrolytic analyses but also has the potential to easily identify additional structural features such as interflavan bond linkage types. We continue to add new samples to our “library” of well-characterized, purified CTs (from 35 different plants) representing diverse CT structural elements, including those found in many forages. Samples from this library have been used for studies of protein precipitation ability, in vitro ammonia reduction and methane abatement during rumen digestion, and anthelmintic and antibiotic activity.
While CT-containing forages can be grazed, there is also a need for stored forages. We have been working to identify which preservation methods (silage, baleage, or hay) would best allow CT-containing forages to deliver appropriate levels of utilizable protein for dairy and other animal production systems (Sub-objective 2.2). We are using birdsfoot trefoil stands that include the popular commercial variety Norcen, along with experimental germplasm bred for low and high CT content. Material harvested during the 2021 and 2022 growing seasons was preserved via the three methods (silage, baleage, hay) across a range (0 to 12 months) of storage times. The resulting stored forage samples are currently being analyzed for N and protein fractions, fiber, and CT content.
To address poor digestibility of plant cell walls (Sub-objective 3.2) in alfalfa, we are examining the role of sugar nucleotide biosynthetic enzymes in cell wall structure and assembly. We are focusing on an enzyme involved in cell wall sugar interconversions leading to the production of two sugars which make up poorly digested xylans. Two alfalfa genes were identified that are predicted to encode the enzyme. Constructs for both over-expression and silencing of these have been transformed into alfalfa, and several independent transformants for each construct have been identified. In the coming year, these will be analyzed for expression, enzyme activity, and ultimately cell wall characteristics and digestibility. We have also produced the large amounts of the enzyme in Escherichia coli to be used for kinetics studies and producing polyclonal antibodies as a tool for further characterization of the in vivo role of the enzyme.
For Sub-objective 3.3, we are developing and testing various methods for screening alfalfa germplasm for decreased protease activity after harvest. Breakdown of whole proteins eventually leads to nitrogen loss from the dairy production system and release of nitrogen into the environment. Alfalfa is a relatively high protein forage, but the plant’s proteases immediately begin to break down the proteins after harvest. We are preparing to screen alfalfa germplasm for reduced protease activity to decrease post-harvest protein breakdown in this forage. As a first step, we are testing the sensitivity of a universal protease activity assay and working to increase its throughput to screen what may be hundreds of alfalfa lines. We are also assessing assays that detect specific types of protease activity such as acid proteinase, aminopeptidase, and carboxypeptidase. If alfalfa lines with decreased protease activity are identified, they should have a higher proportion of intact proteins after harvest. Thus, we are also testing assays that will quantify the amount of intact protein compared to the amount of smaller peptides and amino acids, the products of protein degradation by proteases.
Volatile organic compound (VOC) emissions from fermented forage components represent a loss of energy in dairy rations and an air quality issue. We are currently evaluating a method for the mitigation of silage VOC emissions by application of aqueous solutions at the feed bunk (Sub-objective 4.1A). Gas chromatography-mass spectrometry (GCMS) profiling revealed high sample emission heterogeneity across replicates and no significant effect for most additive treatments. Initial evidence suggests that oil-based additives may increase VOC emissions from silage.
Altering fermentation products produced during ensiling of forages could prevent losses during fermentation and improve utilization by dairy cattle. Succinate is a non-volatile organic acid of agronomic and industrial utility that is used efficiently in the rumen and could reduce production of the greenhouse gas methane in dairy animals. We are working to select silage microbial communities with high succinate production (Sub-objective 4.1B). Our approach includes both selection from silage microbial isolates as well as screening known succinate-producers from the NRRL culture collection for forage fermentation potential.
There is evidence that silage microbial communities can have beneficial probiotic effects for ruminant animals consuming silage. However, the effects of microbial communities from different silages on the rumen microbiome are difficult to distinguish from the effects of nutritional differences of the silages. To address this, we have created microbially-distinct but nutritionally near-identical corn and alfalfa silages using a library of silage inoculants; commercial and lab-isolated (Sub-objective 4.2). Amplicon sequencing data from in vitro rumen digestions are expected to be completed by the end of the fiscal year.
A collaborative team of researchers from ARS, universities, and industry, has been established to develop a next generation, whole-farm dairy simulation model that will have animal, manure, crop/soil, and feed storage modules (Objective 5). We are responsible for development and testing the crop/soil and feed storage modules. Model development is nearing completion for the full model and existing work is being reviewed with a focus on scalability, quality control, and documentation.
Accomplishments
1. Generation of a hairy vetch reference genome. Use of cover crops can help make agriculture more sustainable. Hairy vetch is a legume species that is used as a cover crop, and can reduce soil erosion, help bees, feed livestock, and supply nitrogen to crops like corn and tomatoes. Some traits in hairy vetch limit its use as a cover crop, such as pod shatter and hard seed. To help plant breeders overcome these challenges, ARS researchers at Madison, Wisconsin, and Clay Center, Nebraska, as well as nongovernmental organization and private sector partners generated a reference genome for hairy vetch. The new reference genome is already facilitating breeding for soft seeded varieties that will increase use of hairy vetch as a cover crop. The work is expected to greatly facilitate marker assisted breeding for other useful traits, as well as work in gene discovery, transcriptomics, and genome structure. Additionally, overcoming technical challenges associated with generating this reference genome required the application and creation of new approaches that will benefit similar genomic research on other important species.
2. Two-dimensional nuclear magnetic resonance spectroscopy (2D NMR) is a more facile approach for determining accurate condensed tannin (CT) structural information compared to chemical methods. Condensed tannins (CTs) are a family of chemical compounds present in many plants, including forage crops, that are made up of smaller subunits that can be assembled in many different ways to form CTs of highly diverse structure. CT structure is known to influence important bioactivities that these compounds have such as improving protein utilization and mitigating methane emissions in ruminant animals. ARS researchers in Madison, Wisconsin, have developed new less laborious methods to determine the composition, structure, and purity of CTs using 2D-NMR. Accuracy of the NMR method has been validated by comparison of results to those obtained through labor-intensive traditional wet chemistry approaches. Thus, these improved NMR methods for CT structure determination should find widespread use in identifying CTs responsible for desired biological activities such as improved protein utilization and mitigation of methane emissions in ruminant production systems.
Review Publications
Sullivan, M.L. 2022. Preparation of hydroxycinnamoyl-coenzyme A thioesters using recombinant 4-coumarate:coenzyme A ligase (4CL) for characterization of BAHD hydroxycinnamoyltransferases enzyme activities. In: Jez, Joseph, editor. Methods in Enzymology. Volume 683. Cambridge, MA: Academic Press. p.3-18.
Sullivan, M.L. 2022. Near-real time determination of BAHD acyl-coenzyme A transferase reaction rates and kinetic parameters using Ellman's reagent. In: Jez, Joseph, editor. Methods in Enzymology. Volume 683. Cambridge, MA: Academic Press. p.19-39.
Fanelli, A., Sullivan, M.L. 2022. Bioinformatic tools for protein structure prediction and for molecular docking applied to enzyme active site analysis. In: Jez, Joseph, editor. Methods in Enzymology. Volume 683. Cambridge, MA: Academic Press. p.41-79.
Panke-Buisse, K. 2023. Estimation of silage VOC emission impacts of surface-applied additives by GC-MS. Atmospheric Environment: X.17.Article 100206. https://doi.org/10.1016/j.aeaoa.2023.100206.
Woolsey, I.D., Zeller, W.E., Blomstrand, B.M., Oines, O., Enemark, H.L. 2022. Effects of selected condensed tannins on Cryptosporidium parvum growth and proliferation in HCT-8 cell cultures. Experimental Parasitology. 241. Article 108353. https://doi.org/10.1016/j.exppara.2022.108353.
Dinkins, R.D., Hancock, J.A., Bickhart, D.M., Sullivan, M.L., Zhu, H. 2022. Expression and variation of the genes involved in rhizobium nodulation in red clover. Plants. 11(21). Article 2888. https://doi.org/10.3390/plants11212888.
